Assessment of cervical cells

ABSTRACT

A method of determining premalignant or neoplastic disease state in a tissue sample containing cells of the cervix, the method comprising contacting specific monoclonal antibodies to a cervical tissue sample, determining binding of the antibodies to the sample and comparing the binding with a pattern of binding of the same antibodies to a normal cervical cell sample. Hybridomas which produce suitable antibodies have been deposited at the European Collection of Animal Cell Cultures (ECACC) under the accession numbers ECACC 95020718, 95020716, 95020720, 95020717 and 95020719.

The present invention relates to assessment of cells in a sample oftissue containing cells of the cervix. More particularly, it relates toevaluation of the state of cells of the cervix, discriminating betweennormality and some deviation from normality, and is generally for use inscreening women to detect those whose cervical cells are abnormal.Samples found to be abnormal may be examined in more detail and thecondition of cells in the cervix investigated further. Identification ofa malignant or pre-malignant condition is typically followed byappropriate treatment following more extensive diagnostic procedures.

Cancer of the cervix is the second most common cancer in women. Thecurrent method of detection is the Papanicolau or PAP test, which usesconventional cytological dyes to stain cells in a smear sample, enablingthe visual detection of cellular nuclei and cytoplasm with a microscope.Trained personnel make semi-subjective assessments of the normality orotherwise of the cells examined. The PAP test, though universallyaccepted, is labour-intensive and prone to human error, as evidenced bya number of recent well-publicised scares casting doubt on the accuracyof assessments made by a few of those people who spend their entire daylooking at smear samples.

An alternative or more objective way of assessing the state of cells incervical cell samples would be useful and advantageous. Benefit would beobtained by the removal or at least amelioration of problems arisingfrom the need for subjective, visual evaluation.

It is known that cells within tissues of the mammalian body expresscellular markers that are either unique or partially restricted toparticular cell populations. Different cell populations may bedistinguished by virtue of their individual cell markers. Thus, a cellmay be shown to belong to a particular cell population (eg lymphoidcells) because of its expression of markers defining that population.

These cell markers may be various kinds of molecules, includingproteins, lipids, carbohydrates and combinations of these, such asglycoproteins, glycolipids and lipoproteins.

It is possible to detect cell markers using binding molecules, such asantibodies, with the requisite specificity. Such binding molecules canbe used in qualitative or quantitative detection of cells which bearparticular markers and are thus included in a particular cellpopulation.

Holmes et al have described previously a monoclonal antibody able tobind specifically to an antigen on normal hepatocytes within the liverbut not able to bind cells other than hepatocytes. No binding could bedetected on a number of transplanted and primarydimethylaminoazobenzene-induced hepatomas nor on liver cells frompatients with a variety of liver diseases. (See: Tumour ProgressionMarkers—Proceedings of the Sixth Meeting of the European Association forCancer Research, Budapest 12-15 Oct. 12-15, 1981, 471-481 (1982); Liver(1983), 3: 295-302; Int. J. Cancer (1982), 29: 559-565; Cancer Research(1984), 44: 1611-1624.)

The present invention is founded in the realisation that it is possibleto recognise a pattern of surface antigens on cells of the cervix whichrepresents normality, such that a deviation from a determined and notedpattern of normality can be perceived. Conveniently, antibodies or otherspecific binding molecules may be used in the qualitative and/orquantitative detection of marker antigens on the cells, enablingincreased or reduced expression or loss of one or more of the markers tobe correlated with a disease (or pre-disease) state. In screening, thisenables samples with some deviation from normality to be identified andexamined further, ie suspect samples are highlighted for furtherexamination by suitably qualified personnel. If abnormality in aparticular sample is serious or potentially serious, appropriate stepsmay be taken to examine and then perhaps treat the woman from whom thesample was obtained. Diagnosis and decisions on the need for and natureof treatment remain the domain of clinicians.

For operation of the present invention it is not necessary for themarker antigens actually employed in the test to be identified.Ultimately, what is important is that a panel of antibodies or otherbinding molecules is identified as able to bind cervical cells with apattern which is associated with the cervix being normal, and thatdeviation of binding of those binding molecules from the pattern ofnormality is correlated with the onset of pathological conditions. Theonset of pathology may then be identified in samples containing cervicalcells by some deviation from the established pattern of normality forthose particular binding molecules.

This is exemplified by data included herein relating to five monoclonalantibodies able to bind to various cells of the cervix. The pattern ofbinding of these antibodies to an abnormal tissue sample containingcervical cells is different from the binding to normal cervical samplesenabling identification of abnormality. Clearly, other specific bindingmolecules may be employed successfully in aspects of the presentinvention as long as the requirements set out in the preceding paragraphare satisfied. Such other molecules may bind the antigens bound by theexemplified antibodies, at the same or different epitopes. Indeed theymay bind different antigens altogether.

According to a first aspect of the present invention there is provided amethod of determining abnormality in a tissue sample containing cells ofthe cervix, the method comprising determining binding of antibodies tothe sample and comparing the binding with the pattern of binding of saidantibodies to a normal cervical cell sample. The pattern of binding ofthe antibodies to normal cervical cells may, and generally will, beestablished prior to performance of the method.

The reactivities of antibodies on normal and test samples may bedetermined by any appropriate means. Tagging with individual reportermolecules is one possibility. The reporter molecules may directly orindirectly generate detectable, and preferably measurable, signals. Thelinkage of reporter molecules may be directly or indirectly, covalently,eg via a peptide bond or non-covalently. Linkage via a peptide bond maybe as a result of recombinant expression of a gene fusion encodingantibody and reporter molecule.

One favoured mode is by covalent linkage of each antibody with anindividual fluorochrome, phosphor or laser dye with spectrally isolatedabsorption or emission characteristics. Suitable fluorochromes includefluorescein, rhodamine, phycoerythrin and TEXAS RED®. Suitablechromogenic dyes include diaminobenzidine.

Other reporters include macromolecular colloidal particles orparticulate material such as latex beads that are coloured, magnetic orparamagnetic, and biologically or chemically active agents that candirectly or indirectly cause detectable signals to be visually observed,electronically detected or otherwise recorded. These molecules may beenzymes which catalyse reactions that develop or change colours or causechanges in electrical properties, for example. They may be molecularlyexcitable, such that electronic transitions between energy states resultin characteristic spectral absorptions or emissions. They may includechemical entities used in conjunction with biosensors. Biotin/avidin orbiotin/streptavidin and alkaline phosphatase detection systems may beemployed.

The mode of determining binding is not a feature of the presentinvention and those skilled in the art are able to choose a suitablemode according to their preference and general knowledge.

The signals generated by individual antibody-reporter conjugates may beused to derive quantifiable absolute or relative data of the relevantantibody binding in normal and test samples. In addition, a generalnuclear stain such as propidium iodide may be used to enumerate thetotal cell population in a sampled smear, allowing the provision ofquantitative ratios of individual cell populations relative to the totalcells in a smear, at least where individual antibody reactivitiescorrelate with particular cervical cell populations.

An actual expansion or reduction in the absolute numbers of a particularcell population is not a necessary pre-requisite for the purposes ofthis invention. Any change in the detection of antibody binding (and byimplication change in the phenotypic cell markers) that is detectable,and preferably quantifiable, relative to established parameters ofnormality, is of relevance.

In addition, non-epithelial cells such as leukocytes are known toinfiltrate cervical tissue as a consequence of pathological conditions.These may be enumerated by use of readily available monoclonalantibodies against pan-leukocytic markers, thus providing a furtherlevel of analysis.

Antibodies which are specific for a target of interest may be obtainedusing techniques which are standard in the art. Methods of producingantibodies include immunising a mammal (eg mouse, rat, rabbit, horse,goat, sheep or monkey) with the protein or a fragment thereof or a cellor virus which expresses the protein or fragment. Immunisation with DNAencoding the target polypeptide is also possible. Antibodies may beobtained from immunised animals using any of a variety of techniquesknown in the art, and screened, preferably using binding of antibody toantigen of interest. For instance, Western blotting techniques orimmunoprecipitation may be used (Armitage et al, 1992, Nature 357:80-82).

The production of monoclonal antibodies is well established in the art.Monoclonal antibodies can be subjected to the techniques of recombinantDNA technology to produce other antibodies or chimeric molecules whichretain the specificity of the original antibody. Such techniques mayinvolve introducing DNA encoding the immunoglobulin variable region, orthe complementarity determining regions (CDRs), of an antibody to theconstant regions, or constant regions plus framework regions, of adifferent immunoglobulin. See, for instance, EP184187A, GB 2188638A orEP-A-0239400. A hybridoma producing a monoclonal antibody may be subjectto genetic mutation or other changes, which may or may not alter thebinding specificity of antibodies produced.

As an alternative or supplement to immunising a mammal with a peptide,an antibody specific for a target may be obtained from a recombinantlyproduced library of expressed immunoglobulin variable domains, eg usinglambda bacteriophage or filamentous bacteriophage which displayfunctional immunoglobulin binding domains on their surfaces; forinstance see WO92/01047. The library may be naive, that is constructedfrom sequences obtained from an organism which has not been immunisedwith the target or may be one constructed using sequences obtained froman organism which has been exposed to the antigen of interest (or afragment thereof).

Antibodies may be modified in a number of ways. Indeed the term“antibody” should be construed as covering any specific bindingsubstance having a binding domain with the required specificity. Thusthis covers antibody fragments, derivatives, functional equivalents andhomologues of antibodies, including any polypeptide comprising animmunoglobulin binding domain, whether natural or synthetic. Chimaericmolecules comprising an immunoglobulin binding domain, or equivalent,fused to another polypeptide are therefore included. Cloning andexpression of chimaeric antibodies are described in EP-A-0120694 andEP-A-0125023.

It has been shown that the function of binding antigens can be performedby fragments of a whole antibody. Example binding fragments are (i) theFab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fdfragment consisting of the VH and CH1 domains; (iii) the Fv fragmentconsisting of the VL and VH domains of a single antibody; (iv) the dAbfragment (Ward, E. S. et al., Nature 341, 544-546 (1989)) which consistsof a VH domain; (v) isolated CDR regions; (vi) F(ab′)2 fragments, abivalent fragment comprising two linked Fab fragments (vii) single chainFv molecules (scFv), wherein a VH domain and a VL domain are linked by apeptide linker which allows the two domains to associate to form anantigen binding site (Bird et al, Science, 242, 423-426, 1988; Huston etal, PNAS USA, 85, 5879-5883, 1988); (viii) bispecific single chain Fvdimers (PCT/US92/09965) and (ix) “diabodies”, multivalent ormultispecific fragments constructed by gene fusion (WO94/13804; P.Holliger et al Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993).

Hybridomas able to produce monoclonal antibodies of use in the presentinvention have been deposited and represent individual aspects of thepresent invention, as do the monoclonal antibodies themselves. Thus, thepresent invention provides individually each of the hybridomas depositedas ECACC 95020718, 95020716, 95020720, 95020717 and 95020719, andmutants, derivatives and descendants of each of these hybridomas,whether or not able to produce antibody of the same or alteredspecificity.

The present invention also encompasses use of the hybridomas andantibodies obtainable therefrom in the obtention of other antibodies ofuse in the assessment of the state/condition of cervical cells in atissue sample, ie able to bind to an antigen found on the surface of oneor more cell types of the cervix. Such use may involve isolation of theantigen bound by any of the antibodies obtainable from the depositedhybridomas and use of the antigen in raising further antibodies, eg byimmunisation and/or screening of ‘phage libraries, as discussed above.The antigen may, for example, be isolated by immuno-precipitation fromcervical cell extracts and then used as an immunogen eg for generationof further monoclonal antibodies, or in screening a ‘phage library, asappropriate.

Additionally, nucleic acid encoding one of the antibodies may beisolated from any of the hybridomas and used in a recombinant expressionsystem to produce whole antibody, an antibody fragment or chimaeras ofantibody/antibody fragment fused to another polypeptide (eg label suchas a peptide tag or enzyme). As mentioned above, cloning and expressionof chimaeric antibodies are described in EP-A-0120694 and EP-A-0125023.

Recombinant expression of polypeptides, including antibodies andantibody fragments, is well-known in the art.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known. Suitable host cells includebacteria, mammalian cells, yeast and baculovirus systems. Mammalian celllines available in the art for expression of a heterologous polypeptideinclude Chinese hamster ovary cells, HeLa cells, baby hamster kidneycells and many others. A common, preferred bacterial host is E. coli.

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorfragments, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. For further details see, forexample, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrooket al, 1989, Cold Spring Harbor Laboratory Press. Transformationprocedures depend on the host used, but are well known.

Thus, the present invention extends to any antibody or antibody fragmentable to bind an antigen to which any (ie one or more) of the antibodiesobtainable from the deposited hybridomas is able to bind. Ability tobind the same antigen may be assessed, for example in a bindinginhibition assay or in a band-shift assay on an electrophoretic gel.

The present invention also provides in a further aspect the use of anyantibody or antibody fragment of the invention, including thoseobtainable from any of the deposited hybridomas, in assessment of thenature or condition of cells of the cervix in a tissue sample, asdisclosed.

Further aspects and embodiments of the present invention will beapparent to those skilled in the art.

Before illustrating embodiments of the present invention in more detailby way of example, it is helpful to understand the organisation of thecervix and the relationships between the different cell-types foundthere.

Cell Populations of the Cervix in Normal and Pathological Conditions

The cervix is essentially composed of two distinct cell types: thesquamous epithelium and the columnar epithelium, each of which islocated in an anatomically distinct region of the tissue. The squamousepithelium is located at the exterior aspect (the exocervix) of thecervical opening (the cervical os), while the columnar epitheliumextends into the endocervical canal (the endocervix). These two distinctepithelial cell types come into contact in the vicinity of the cervicalos, at the squamo-columnar junction. The squamo-columnar junction is ofclinical importance as it is the region where the majority ofmalignancies arise. For diagnostic validity, a cervical smear samplemust include cells from this region. In order to ensure that this hasbeen achieved, a smear must contain columnar as well as squamousepithelial cells.

Columnar cells are the source of cervical mucous. They are generallyarranged as a single cell layer lining the endocervix, which is throwninto deep folds to form the cervical glands. A small proportion (5%) ofcervical tumours are derived from columnar cells i.e. theadenocarcinomas.

In some cervices, a cuboidal cell layer, the so-called reserve cellpopulation, is disposed beneath the columnar cells. The role of reservecells is unclear, but believed by many investigators to give rise tocolumnar epithelium.

In contrast to the columnar epithelium, the squamous epithelium, fromwhich most (95%) cervical tumours arise, is a multilayered dynamic stemcell system under constant renewal.

The stem cell compartment itself is located adjacent to the basementmembrane within the basal cell layer. Stem cell division gives rise toparabasal, intermediate, and superficial cell derivatives. These areconventionally defined in terms of both their characteristic morphologyand location within the squamous epithelium. The transition from basalcells located in the deepest layer of the squamous epithelium, tosuperficial cells at its surface is associated with progressivedifferentiation and a loss of proliferation until superficial squamousepithelial cells at the cervical surface are terminally differentiated.

The transition zone (TZ), adjacent to the squamo-columnar-junction is ofclinical importance, as it includes a region of metaplasticsquamous-epithelium. This is generated at puberty, in response to theacidic environment of the vagina. The data presented herein quantify theproportion of samples where the TZ is present.

EXEMPLIFICATION OF EMBODIMENTS OF THE PRESENT INVENTION BY WAY OFILLUSTRATION AND NOT LIMITATION

Abbreviations

ATCC—American Type Culture Collection; CIN—Cervical intra-epithelialneoplasia; CHAPS(3-[Cholamidopropyl)-dimethylammonio]-1-propanesulfonate); CD—ClusterDesignation nomenclature of established cell markers; DAB—Di-aminobenzidine; ECACC—European Collection of Animal Cell Cultures;EDTA—Ethylene di-amine tetra acetic acid; HLA—Human Leukocyte Antigen;HRPO—Horse radish peroxidase; Ig—Immunoglobulin; kDA—Kilo dalton;Mab(s)—Monoclonal antibody(ies); Mwt—Molecular weight; NS1—Non-secretor1; PAGE—Polyacrylamide gel electrophoresis; PBS—Phosphate bufferedsaline; PAP—Papanicolau test; SDS—Sodium dodecyl sulphate; TBS—Trisbuffered saline; TZ—Transition zone; W/V—Weight/volume ratio.

Cell Markers

The work disclosed herein shows that under normal conditions, cellsurface markers may be gained or lost as cells within a given lineageproceed along their differentiation pathway. Therefore, monoclonalantibodies or other binding molecules exhibiting specific reactivitiesagainst these markers provide a means of monitoring the normalprogression of cells along their differentiation pathway. Inpathological conditions, the normal expansion or loss of these markersmay be perturbed. Consequently, an expansion or loss of a cell surfacemarker is detectable as a particular cell population proliferates orbecomes arrested at a discrete stage in its differentiation pathway.

The antibody reactivities described herein illustrate the situationwhere an overlapping continuum of reactivities is observed throughoutnormal squamous epithelial cell differentiation in the human cervix.

The designated nomenclature of the five monoclonal antibodiesexemplified herein is:

-   1. CV3.6B5/F3/C2, hybridoma deposited as ECACC 95020718-   2. 2C7/B4/D6., hybridoma deposited as ECACC 95020716-   3. CV5.9G5.C6, hybridoma deposited as ECACC 95020720-   4. HG3/E11/C4, hybridoma deposited as ECACC 95020717-   5. BC4/E7/E5, hybridoma deposited as ECACC 95020719    Generally herein, these antibodies are referred to in their    abbreviated forms. The hybridomas were deposited at European    Collection of Animal Cell Cultures (ECACC), Centre for Applied    Microbiology & Research, Salisbury, Wiltshire SP4 0JG, United    Kingdom on Feb. 6, 1995.

Thus:

Basal cells are 6B5+ BC4− 9G5− HG3− 2C7− Parabasal cells are 6B5+ BC4+9G5− HG3− 2C7− Intermediate cells are 6B5− BC4+ 9G5+ HG3+ 2C7−Superficial squames are 6B5− BC4− 9G5+ HG3+ 2C7− Columnar cells are 6B5+BC4− 9G5− HG3− 2C7+.

These characteristic antibody reactivity profiles reproducibly definediscrete stages in the differentiation of squamous epithelial cells.Additionally, they readily allow columnar epithelial cells to bedistinguished from squamous epithelial cells.

Brief Description of Results

Immunoprecipitation of the 6B5 target protein from the cell surface of acervical carcinoma cell line and from membrane preparations of cervicalsquamous epithelium:

-   -   (a) Cell surface proteins on the cervical carcinoma cell line        C4II (Auersperg 1969. J. Natl. Cancer Inst. USA 43 151-173) were        radioiodinated by the lactoperoxidase method (Marchalonis 1969.        Biochem J. 113 299-305). Cells were solubilised with the        detergent CHAPS        (3-[3-Cholamidopropyl)-dimethylammonio]-1-propanesulfonate).        Immunoprecipitations were carried out with test Mab against        equal volumes of lysate (Houlihan et. al. 1992 J. Immunol. 149        668-675). The results show that Mab 6B5 detects a cell surface        dimeric protein with components of approx. 181 and 184 kDa in a        cervical epithelial cell line.    -   (b) Standard membrane extracts were prepared from sheets of        cervical squamous epithelium (Evans 1979. Laboratory techniques        in biochemistry and molecular biology, Eds. Work and Work 7        1-266, Elsevier). Membranes were radioiodinated using Iodobeads        (Markwell 1982. Anlyt. Biochem. 125 427-432) and solubilised in        CHAPS. Immunoprecipitations were carried out as described above.        The results show that a similar form of this protein is also        present in a membrane-associated fraction of cervical squamous        epithelium shown in (a).

Immunoprecipitated material was analysed by SDS-PAGE under reducingconditions and autoradiography (Laemmli 1970. Nature 227 133-681).

Affinity Isolation of the 6B5 Target from C4II Cells

Affinity chromatography was performed as described by Houlihan et al.1992 (J. Immunol. 149 668-675). Purified 6B5 antibody was conjugated toprotein G-sepharose and cross-linked with dimethyl pimelimidate. 2×10⁸C4II cells were solubilised in the detergent CHAPS. The lysate waspassed through a preclearing column containing mouse Ig-sepharose andprotein A sepharose (Sigma) and then through the 6B5 antibody column.Both columns were washed with 10 bed volumes of lysis buffer and theneluted with 50 mM triethylamine, pH 11.5. The eluate was neutralisedwith 2 M glycine pH 2.0, concentrated by microultrafiltration andanalysed by SDS-PAGE under reducing conditions and coomassie bluestaining.

A single 180 kDa product was specifically isolated by the 6B5 column anddetected in reduced eluates. This component was not observed in theeluate representing the negative control—mouse Ig/protein A sepharosecolumn. Immunoglobulin H-chain which had leached during elution from thenegative control column was present, however, in the mouse Ig/protein Asepharose eluate.

Immunoblotting of Detergent-Solubilised Endocervical Material usingAntibody 2C7

Endocervical tissue was minced in Hanks buffered saline and proteinswere solubilised in CHAPS(3-[3-Cholamidopropyl)-dimethylammonio]-1-propanesulfonate) buffer. Thedetergent soluble material was concentrated by microultrafiltrationusing a 30 kD cut-off filter. Proteins were electrophoreticallyseparated on 1% agarose gels in Laemmli sample buffer under reducingconditions. Proteins were transferred to microporous membranes forimmunoblotting. Identical strips were probed with Mab 2C7 and tissueculture supernatant as a negative control. Parallel strips were stainedwith periodic acid-Schiffs reagent which stains glycoproteins, andcoomassie blue as a general protein stain (Methods were based on thoseof Morales et. al. 1993 Human Reproduction 8 78-83).-ve controlimmunoblot employed tissue culture supernatant.

Mab 2C7 specifically detects components migrating in the high m.wt.range that correspond to material stained by periodic acid-Schiff'sreagent. The large m.wt (>500 kDa) of this material and detection byperiodic acid-Schiff's reagent is consistent with the presence ofmucins. The Mab showed no reactivity with any proteins in thesignificantly lower m.wt. material stained by coomassie blue.

Immunoblotting of Detergent-Solublised Cervical Epithelium using Mab 9G5

Squamous epithelial sheets were isolated from normal cervices using theenzyme Dispase (Boehringer). Sheets were solubilised in the detergentCHAPS (3-[3-Cholamidopropyl)-dimethylammonio]-1-propanesulfonate).Cytokeratin-enriched extracts were prepared from the CHAPS-insolublematerial by the method of Franke et al. 1981 (Exp. Cell Res. 131209-213). Both detergent (D) and cytokeratin (C) extracts wereelectrophoretically-separated by SDS-PAGE under reducing conditions(Laemmli 1970 Nature 227 133-681) and proteins were transferred tomicroporous membranes for immunoblotting (Towbin et al. 1979. Proc.Natl. Acad Sci USA 76 4350-4354).

Mab 9G5 detects a single 40 kDa product in detergent-solubilisedextracts but not in cytokeratin extracts of cervical squamousepithelium. A pan-reactive cytokeratin Mab AH3 (J. M. Houlihan Ph.D.thesis University of Bristol 1993) was used as a control; keratins aredetected in the cytokeratin extract.

Affinity Chromatographic Isolation of the 9G5 Target Protein from HumanAmnion Cells

Affinity chromatography was performed as described by Houlihan et al.1992 (J. Immunol. 149 668-675). Purified Mab 9G5 was conjugated toprotein G-sepharose and cross-linked with dimethyl pimelimidate. 2×10⁸amnion cells were isolated from the term placental membranes byincubation in trypsin followed by collagenase/hyaluronidase by themethod of Holmes et al. 1990. (J. Immunol. 144 3099-3015) andsolubilised in the detergent TX-100. The lysate was passed throughpreclearing columns, comprising mouse Ig and protein A sepharose andthen the Mab 9G5 column. The Mab 9G5 column was eluted with 50 mMtriethylamine, pH 11.5. The eluate was concentrated bymicroultrafiltration and analysed by SDS-PAGE under non-reducingconditions and coomassie blue staining.

A single 40 kDa product was isolated from amnion cell lysates by the Mab9G5 affinity column. A fraction of this eluate was examined byimmunoblotting with the 9G5 Mab and an irrelvant IgG1 Mab as a negativecontrol. The 9G5 reacted with the 40 kDa product. The strip probed withthe negative control Mab was blank.

Immunoblotting of Detergent-Solubilised Cervical Epithelium using MabHG3

Squamous epithelial sheets were isolated from normal cervices using theenzyme Dispase (Boehringer). Sheets were solubilised in the detergentCHAPS (3-[3-Cholamidopropyl)-dimethylammonio]-1-propanesulfonate). Equalamounts of the CHAPS-soluble material were electrophoretically-separatedby SDS-PAGE (Laemmli 1970. Nature 227 133-681) under both non-reducingand reducing conditions. Proteins were transferred to microporousmembranes for immunoblotting (Towbin et al. 1979. Proc. Natl. Acad SciUSA 76 4350-4354). Parallel strips tested with Mab HG3; tissue culturesupernatant was used as a negative control.

The negative control blot was blank.

(NR—non-reduced; R—Reduced)

Mab HG3 detects a product of approx. 180 kDa, under both non-reducingand reducing conditions, in detergent soluble cervical squamousepithelium.

Immunoblotting of Detergent-Soluble and Detergent-Insoluble CervicalEpithelium using Mab BC4

Squamous epithelial sheets were isolated from normal cervices using theenzyme Dispase (Boehringer). Sheets were solubilised in the detergentCHAPS (3-[3-Cholamidopropyl)-dimethylammonio]-1-propanesulfonate).CHAPS-soluble and CHAPS-insoluble material was adjusted to the samevolume. Equal amounts of both soluble and insoluble fractions wereelectrophoretically-separated by SDS-PAGE (Laemmli 1970. Nature 227133-681). The detergent soluble fraction was analysed under bothnon-reducing and reducing conditions while the detergent insolublefraction was examined under reducing conditions. Proteins weretransferred to microporous membranes for immunoblotting (Towbin et al.1979. Proc. Natl. Acad Sci USA 76 4350-4354). Parallel strips weretested with Mab BC4; tissue culture supernatant was used as a negativecontrol.

The negative control blot was blank.

Mab BC4 detects a protein of 200-210 kDa in non-reduced, but not inreduced, detergent soluble extracts of cervical squamous epithelium. Noproduct was detected in detergent insoluble fraction of cervicalsquamous epithelium.

Cervical Biopsies

Most of the data herein has been obtained from extensive investigationsundertaken on biopsy material, for the following reasons:

(a) A single biopsy specimen provides several serial tissue sections(each 5 um thick) for microscopic examination. All sections aretherefore almost identical, and the various cell populations located inthe same positions relative to each other. Similarity in anatomicalstructure, at the cellular level, is therefore maintained betweensamples.

This enables reactivities of different antibodies to be investigated andcompared, on the same cell populations, between samples.

Different cell populations within a tissue are identified by theirindividual morphologies and locations. Therefore, the reactivities ofindividual antibodies against such cells in a biopsy sample, enablestheir specificities to be determined and established.

(b) Pathological changes are accompanied by a disruption of the normalhistology of the tissue. Therefore, antibody reactivities can only beeffectively investigated (and correlated with pathological conditions)in samples, where the tissue architecture is representative of thesituation in vivo.

Pathological changes that precede malignancy are classified according toan established system of grading: CIN I, II and III. The progression ofdisease leads to carcinoma in situ and finally a frank tumour.

(c) Most importantly, the use of biopsy material was necessary todetermine whether modulation of expression of the antibody targetsoccurred as a function of disease in the cervix. The profiles ofantibody reactivities on tissue sections of normal biopsies provided aframework to establish their corresponding reactivities on abnormalsmears. Such reactivities on normal and abnormal biopsies enabled theselection of antibodies for use in a cervical smear screening system, inaccordance with the present invention.

Cervical Smears

The cells in a smear sample do not maintain their topographicalrelationships to each other, as in a tissue section of a biopsy.Antibodies selected on the basis of their specificities against variouscell populations (as above), were investigated for their ability toprovide qualitative or quantitative information on normal andpathological smear samples. Their absolute or relative numbers,enumerated with these antibodies, provides a means of determiningdetectable changes in these cell populations.

Tissue Distribution

Although the monoclonal antibodies were raised against cervicalepithelium, they would be expected to react against their targetepitopes if present on non-cervical epithelium of shared embryologicalorigin. Therefore, their tissue distribution in other epithelial tissuewas determined, enabling further characterisation at that level.

Biochemical Data

These have been mainly derived using extracts of cervical amnion andplacental tissue, electrophoretically separated on SDS-PAGE gels underreducing or non-reducing conditions. Western blots of the resolvedcomponents were then probed with the panel of monoclonal antibodies,enabling their individual target epitopes to be defined at the molecularlevel.

The detection of such resolved components under defined experimentalconditions provides information on the molecular structure of the targetantigen. If detected under both reducing and non-reducing conditions, itcan be deduced to be a non-conformationally dependent monomericstructure. On the other hand, if only detected under non-reducingconditions, the epitope is present on a conformationally dependentstructure.

The methods of extraction enable the identified material to be assignedto known classes of cell markers. For example, use of detergents do notprovide extracts with appreciable amounts of cytokeratins. Thecytokeratins are a complex family of cytoplasmic filamentous proteinstructures that are well documented in the scientific literature. Theyare biochemically and antigenically related to varying degrees, and areexpressed in different epithelia in different combinations ofpolypeptides. A given epithelium or epithelial cell can therefore becharacterized by the specific pattern of its cytokeratin components[Ref: Moll R et al, 1982].

Furthermore, with the exception of Mab 9G5, the molecular weights of thetarget antigens precluded them from belonging to the cytokeratin family.A monoclonal antibody (AH3) with pan-Cytokeratin reactivity was used toconfirm that the 9G5 epitope was a non-cytokeratin cell marker. Thisantibody has been previously described [Ref: Houlihan 1993].

Where sufficient or suitable biopsy material was not obtainable,established carcinoma cell lines derived from cervical tissue wereutilised to prepare such extracts.

Trypsin Sensitivity

The susceptibility of target epitopes to proteolysis by trypsin wasdetermined. The enzyme's pronounced substrate specificity (restricted tolysine and arginine residues) provides a means of characterising thetarget epitopes further.

Materials and Methods

Standard buffers and reagents were prepared in accordance withestablished procedures that are well documented in the scientificliterature so need not be detailed here. Unless otherwise stated, allchemicals were obtained from Sigma Chemical Co, Poole, Dorset, UK.

Preparation of Immunogen and Immunisation

Three sources of cervical cells: (A) smear samples, (B) hysterectomiesand (C) premalignant CIN biopsies were evaluated for their potential useas immunogen:

(A)

Cervical smears from routine samples were dispensed into sterile PBS andwashed twice. The yield, composition and viability of cells in smearstaken from different sites within the cervix was examined. The cellularcomposition of conventional smears taken from the exocervix using aspatula, was compared with endocervical smears taken with a brush.Yields of squamous cells in exocervical smears varied; obtaining up to10⁶, with viabilities of 50-60%. Endocervical smears gave much loweryields of typically 10⁴, which also contained predominantly squamouscells.

Cytospin preparations of cervical smear samples were also examinedimmunohistologically and identified using anti-cytokeratin antibodies.The overwhelming majority were squames; the preparations contained fewbasal, parabasal, or columnar epithelial cells. It was concluded thatcervical smears did not represent an effective source of material foruse as immunogen, since they did not contain a sufficient cross sectionof cervical epithelial cell populations.

(B)

Total hysterectomy specimens are typically obtained from women withmenorrhagia or fibroids; in these, the cervix is essentially normal.Such specimens are typically in the perimenopausal (38-45 yr old)period. The optimal method for obtaining single cell suspensions fromwhole normal cervical biopsy material was determined to be as follows.

The excess stromal tissue was removed with a scalpel. The fragments oftissue were floated in a solution of the enzyme Dispase II (1.2units/ml, Boehringer Mannheim, Sussex, UK) in HANKS buffered salinelacking Ca²⁺/Mg²⁺ overnight at 4° C. This procedure disrupted theepithelial/stromal junction, such that sheets of epithelial cells couldbe gently teased apart. They were washed by low speed centrifugation inHANKS buffered saline lacking Ca²⁺/Mg²⁺ and resuspended in 0.05%trypsin/0.02% EDTA (both w/v). After a 30 minute incubation at 37° C.with stirring, trypsinization was arrested by the addition of 1.3 mg (in5 ml of saline) of soya bean trypsin inhibitor.

The secondary enzyme digestion with trypsin disaggregated the epithelialsheets to generate a single cell suspension.

Large fragments were allowed to settle out under gravity and thesupernatant was removed. The cell suspension was drawn through a 16gauge needle to break up cell clumps, and filtered through a 100 umgauze. Cells were washed twice in PBS prior to use as immunogen.

(C)

In addition to normal cervical biopsies, immunogen from pathological CINII/III biopsies were also used. The material was treated similarly,except for the initial overnight enzyme treatment also being in trypsin.

The immunisation regime consisted of an initial intra-peritonealinoculation of 5×10⁵ cells as a priming dose, followed by five furtherimmunisations of 0.5-2×10⁶ normal epithelial cells, at 2-3 weeklyintervals in 8 week old female Balb/c mice.

As some proteolytic cleavage of cell surface components was possible,the effect of enzyme treatment on the integrity of epithelial cellsurface markers was ascertained. This was determined by monitoring itseffect on CD44, CD55 and HLA Class 1 antigens, all of which areexpressed on cervical epithelial cells. These established cell surfacemarkers can be detected by appropriate monoclonal antibodies which arereadily commercially available [Ref: Knapp W, 1989]. These markers werefound to be lost or reduced as a consequence of prolonged exposure totrypsin at 37° C. However, adopting measures to minimise such effectsi.e. reduced exposure at 37° C. and use of the enzyme dispase in theinitial overnight incubation at 4° C. instead, ensured that thephenotypic profile of the disaggregated cells was maintained veryclosely to that of native cervical epithelium.

Fusion and Assay of Supernatants from Hybridomas

Splenocytes from the immunised mice were fused with NS1 murine myelomacells, and hybrids selected by conventional methodology that isdescribed in a number of publications [Ref: Kennet R et al, 1980, andSchrier M et al, 1980].

Candidate monoclonal antibodies were selected on the basis of theirreactivities on tissue sections of cervical biopsies using establishedimmunohistological techniques [Ref: Holmes C H et al, 1990].

Briefly, sections were cut from frozen tissue blocks in a cryostat at 5μm thickness, thawed, air dried at room temperature for 1 hour, fixed inice-cold acetone for 10 mins and immunostained by an indirectimmunoperoxidase technique. Supernatants, from wells containing growinghybridomas, were incubated for 45 mins at room temperature on tissuesections. After washing in TBS for 5 minutes, they were incubated for 30mins with a commercially available HRPO-conjugated rabbit anti-mouse Igreagent, optimally diluted in TBS containing 10% normal human serum.After two more washes, sections were developed with DAB and hydrogenperoxide for 5 mins. After stopping the reaction by washing the slidesin tap water for 5 mins, the sections were counterstained inhaematoxylin, dehydrated, cleared in Histoclear and mounted in DPXmountant.

Candidate hybridomas, secreting antibodies of interest, were cloned tostability by the method of limiting dilution. The antibodies secreted bysuch clones were reassayed to confirm antibody specificity byimmunostaining, as above.

Ig production by hybridomas was also screened by an ELISA technique,using commercially available reagents from Dako AS, Copenhagen,Denmark): Rabbit anti-mouse Ig (product no: Z259) optimally diluted at{fraction (1/2000)} was used as the solid phase capture reagent.Supernatants from hybridomas were incubated for 60 min; bound antibodywas detected with a HRPO-conjugated rabbit anti-mouse Ig (product no:P260) diluted {fraction (1/1000)} incubated for 45 mins. Bothincubations were at room temperature; the reagents were diluted in PBS,and the wells washed between incubations with PBS-0.025% Tween.

Biochemical Characterisation

The methods utilised are generally well known and documented inpublished laboratory technical manuals [Ref: Harlow E and Lane D, 1988and Work and Work, 1979] and in individual publications [Ref:Marchalonis, 1969; Markwell, 1982; and Laemmli, 1970]. Detaileddescriptions of specific procedures undertaken in these investigationshave been previously published [Ref: Holmes C H et al 1990 and HoulihanJ M et al, 1992].

EXAMPLE 1 Mab 6B5

This antibody of isotype IgG1 was raised against an immunogen preparedfrom squamous epithelial cells isolated from the cervices ofhysterectomy specimens.

Reactivity

Its specificity is mainly against parabasal and basal cells in cervicalsquamous epithelium together with basement membrane. However, it alsocross-reacts with columnar epithelium and non-epithelial stromalelements.

Although parabasal cells are not present in large numbers in normalsmears, the antibody does identify the small number that are present;based cells are usually not present in smear samples. However, 6E5reactivity increases significantly in pathological conditions. Withtumours, 6/7 squamous cell carcinomas showed antibody reactivity. Thisreactivity was retained on one adenocarcinoma; this was expected, as 6B5also reacts with normal columnar cells. There is also an expansion of6B5 reactivity in CINs: in 9/15 moderate to severe CIN specimens tested,there was a marked expansion in the affected squamous epitheliumrelative to the native unaffected epithelium.

The anti-stromal reactivity is only of relevance in biopsy material, asstromal elements are not present in smear samples. The anti-columnarreactivity is not expected to interfere in the intended mode ofapplication, as adequate controls exist to correct for this effect (seereactivity of Mab 2C7).

(a) Normal Cervical Epithelium

No of tests: 90; No of patients: 44 (with TZ: 21)

Parabasal cells and basement membranes were intensely reactive insquamous epithelium. Columnar cells were also positive, with reactivityexpanded in the transformation zone in 21/21 specimens.

(b) Premalignant Cervical Epithelium (CIN II/III or III)

No of tests: 42; No of patients: 30 (with CIN: 15)

The reactivity was expanded in 9/15 CIN specimens.

(c) Cervical Carcinomas

The antibody reacted strongly with 5/7 squamous cell carcinomas and withboth adenocarcinomas tested.

Tissue Distribution

Despite its restricted distribution in the cervix, the target epitope ispresent in other epithelial tissue:

Placenta

Epithelial cells in term placental membranes (amnion andcytotrophoblasts) are positive. The basement membrane beneath thesyncytiotrophoblast is positive. In first trimester placenta, bothvillous cytotrophoblast and syncytiotrophoblast are positive. Theantibody shows differential activity with cytotrophoblast inextravillous cell columns: cells at the base of these columns arepositive while those at the periphery are negative.

Kidney: glomeruli +; tubules − Pancreas: ducts and acini +; laminapropria − Colon: epithelium +; lamina propria − Liver: hepatocytes +;bile duct/mesenchymal cells − Endometrium: glandular epithelium −;lamina propria −; myometrium −; arteries − Epidermis: parabasal cells +;basement membrane + (foreskin) basal cells +/−; intermediate cells −;superficial cells −Biochemical

Because of its limited reactivity in the normal cervix, an establishedcervical carcinoma cell line C4II was used to characterise this antibodybiochemically. The C4II cell line [Ref: Auersperg N and Hauvryl AP, 1962and Auersperg N, 1969] obtained from ECACC, Porton Down, UK is welldocumented in the scientific literature. It is also deposited in theATCC under Accession No. CRL 1595.

Mab 6B5 detects a dimeric product of approximately 181-184 kDa, byimmunoprecipitation on the cell surface of C4II. A single componenthaving this molecular weight has subsequently been immunoprecipitateddirectly from radiolabelled membrane preparations of normal invivo-derived cervical squamous epithelial cells. However, the antibodydoes not detect these components on cervical extracts by westernblotting. Taken together, these data indicate that Mab 6B5 detects aconformationally-dependent epitope on a cell surface (non-cytokeratin)protein. The 6B5 target has also been isolated to a high degree ofpurity by immunoaffinity chromatography, for N-terminal sequencing.

Trypsin Sensitivity

The reactivity of the antibody is maintained with the C4II cell lineafter a short 10 min exposure to trypsin at 0.05% (w/v). However,antibody reactivity with placental membranes or amnion cells isabolished after a longer 1 hr exposure to trypsin at 0.1% (w/v). Theantibody target is therefore only partially resistant to trypsin.

EXAMPLE 2 Mab 2C7

This antibody of isotype IgG1 was raised against an immunogen preparedfrom normal cervices of hysterectomy specimens.

Reactivity

The antibody reacts specifically and solely with columnar epithelialcells.

(a) Normal Cervical Epithelium

No of tests: 83; No of patients: 43 (with TZ: 24)

This antibody reacted specifically with columnar epithelial cells, andnot with any other cell population in the cervix. The squamousepithelium was negative in all cases.

(b) Premalignant Cervical Epithelium (CIN II/III or III)

No of tests: 42; No of patients: 30 (with CIN: 15)

No reactivity with either normal or neoplastic epithelium; only adjacentcolumnar epithelium was stained.

(c) Cervical Carcinomas

The antibody did not react with 7/7 squamous carcinomas tested, asexpected. However, it reacted with one of the two adenocarcinomastested.

It can be concluded that the target epitope of Mab 2C7 is solelyrestricted to columnar cells, and not expressed in either normalsquamous epithelium or in CIN lesions. The antibody is considered usefulsince the presence of columnar cells on a smear indicates that thesquamo-columnar junction has been sampled, and hence that the smear ispatent (or adequate). A correctly sampled smear therefore containsbetween 1-5% of 2C7 reactive cells.

The 2C7 target epitope is distinct from that of 6B5. In normal smears,both antibodies identify the columnar cell population. However, whilst6B5 additionally reacts with parabasal cells, 2C7 only detects columnarcells. Therefore, together, they provide a means of enumerating bothcell populations by analysis of their individual reactivity profiles.

Tissue Distribution Placenta: The amnion and cytotrophoblast inplacental membranes, and syncytiotrophoblast were negative in firsttrimester placentae and at term. Pancreas: Epithelium of ducts + Colon:Glandular epithelium + Tonsil: Stratified epithelium − Epidermis:Foreskin − Liver: Hepatocytes− ; bile duct +/− Endometrium: Epithelium −Biochemical

Detergent extracts of endocervical epithelium were prepared byincubating fragments of endocervix in buffer containing CHAPS. OnWestern blots of material separated by SDS-PAGE, the antibody reactedwith unresolved high Mwt components of >400,000 kDA in these extracts.This was further investigated by resolving the extracted material on 1%agarose gels, which are more suitable than SDS-PAGE gels for largermolecules. The antibody reacted with components that preciselycorresponded to a fraction that also stained with periodic acid-Schiff'sbase, but not with Coomassie Blue. This indicated that the targetepitope represented a mucin or a mucin-associated product.

Trypsin Sensitivity

The target epitope appears to be trypsin insensitive, as the antibodyreacted with cell preparations of cervical tissue which had beentrypsinised for periods of up to 1 hr.

EXAMPLE 3 Mabs 9G5 and HG3

Both antibodies are of isotype IgG1.

Mab 9G5 was raised against an immunogen prepared from squamousepithelial cells isolated from normal cervices of hysterectomyspecimens.

Mab HG3 was raised against an immunogen prepared from cervical biopsiesof pathological CIN material.

Reactivity

Both antibodies react primarly with superficial and intermediatesquamous epithelium in the cervix.

(a) Normal Cervical Epithelium

No of tests: 83; No of patients: 44 (with TZ: 21 for Mab 9G5 and 24 forMab HG3)

Both antibodies have similar patterns of reactivity in the normalcervix, with specificities against superficial and intermediate squamousepithelial cells. They are unreactive with basal cells; however, Mab 9G5reacts with parabasal cells in some specimens, where the reactivityfades within the upper parabasal layer. In contrast, Mab HG3 does notreact with parabasal cells in any of the specimens.

Mab 9G5 does not react with columnar cells; however, Mab HG3 shows weakreactivity on columnar cells in a small number of specimens.

(b) Premalignant Cervical Epithelium (CIN II/III or III)

No of tests: 42; No of patients: 30 (with CIN: 15)

In general, both antibodies show detectable differences between normaland abnormal cervical epithelium. The reactivity is modulated in CINs,where it is either reduced or absent; e.g. the reactivity of Mab 9G5 ismarkedly reduced in 10/15 specimens. In these lesions, the depth ofimmunostaining (denoting antibody reactivity) is reduced in terms of thenumber of cell layers, when compared to normal squamous epithelium.

(c) Cervical Carcinomas

Both antibodies differed in their reactivities against the sevensquamous cell carcinomas tested:

Mab 9G5 was completely unreactive with 3/7 specimens tested. Theremaining 4 tumours showed heterogenous reactivity.

Mab HG3 showed extensive reactivity with 3/7 of the squamous cellcarcinomas, some reactivity with 2, and was unreactive with theremaining 2.

Heterogeneity is defined as areas of both reactivity and unreactivitywithin a particular specimen. Generally, despite similar reactivities inthe normal cervix, the reactivity of Mab 9G5 with these tumours was morelimited than that of Mab HG3. In particular, Mab 9G5 was negative withtwo tumours which Mab HG3 reacted extensively with.

When tested on two adenocarcinomas, Mab HG3 reacted with both tumours;whereas Mab 9G5 was unreactive against both.

Mabs 9G5 and HG3 recognise distinct target epitopes on the same cellpopulations. However, it is considered desirable to utilise them intandem, for the following reasons:

-   (a) It is not known whether all cells that comprise the intermediate    and superficial cell population display both target epitopes. Even    if they normally do, their expression may modulate with stages of    the cell or oestrus cycles.-   (b) As superficial epithelial cells are a mainly dead or dying cell    population, it is conceivable that their surface markers would be    heterogenous in their detectability.-   (c) Cervical smears are largely comprised of superficial and    intermediate cells; with columnar and parabasal cells being a    minority. Consequently, a decisive factor in deriving conclusions    regarding the clinical status of the sample, would be based on the    information concerning these cells.

For these reasons, it may be prudent to avoid dependence on a singleantibody. Therefore, both antibodies may be utilised to enumerate oranalyse absolute or relative numbers of intermediate and superficialsquamous cells in normal or pathological conditions.

Tissue Distribution Kidney, pancreas: Negative liver and endometriumColon: Mab 9G5 − ; Mab HG3 + Tonsil: Stratified epithelium + Epidermis(foreskin): Superficial and intermediate cells + Placenta : Amnion andcytotrophoblast in placental; membranes + Synciotrophoblast +[In the first trimester, these trophoblast populations were eithernegative or only weakly stained.]Biochemical

Mab 9G5 cross-reacts with amnion and placental trophoblast epithelium.On immunoblots, it detects a prominent 40 KDa component under bothreducing and non-reducing conditions, from detergent extracted material.Therefore, the 9G5 target epitope is likely to be on a non-cytokeratinmonomeric protein, which is not conformation dependent. Micro-sequenceanalysis indicates that the N-terminus is blocked by a methionineresidue; further sequence analysis is in progress on proteolytic digestsof the intact molecule. Mab HG3 detects a 180 KDa component underreducing and non-reducing conditions, in both detergent soluble andmembrane preparations of both amnion and cervix. Therefore, its targetepitope is also likely to be a conformationally independent monomericprotein.

EXAMPLE 4 Mab BC4

This antibody of isotype IgM, was raised against epithelial cellsisolated from premalignant cervical biopsies containing CIN.

Reactivity

The antibody primarily reacts with parabasal and intermediate cells inthe cervix.

(a) Normal Cervical Epithelium

No of tests: 85; No of patients: 44 (with TZ: 24) Typically, thereactivity encompasses 2-6 cell layers above the parabasal layer, andtherefore can include the lower layers of intermediate squamous cells.Basal cells, superficial squames and columnar cells are negative.

This reactivity pattern, when compared with that of Mabs 6B5, 9G5 andHG3 (see Table 1), enables the intermediate squamous cell population tobe enumerated by appropriate deduction.

(b) Premalignant Cervical Epithelium (CIN II/III or III)

No of tests: 42; No of patients: 30 (with CIN: 15)

Reactivity on parabasal cells in CIN specimens is either absent orgrossly disrupted in 7/15 specimens. Where there was a complete loss ofreactivity, the loss occurred abruptly at the junction between normaland premalignant tissue. Where the reactivity was reduced, it was interms of the number of immuno-stained cell layers.

(c) Cervical Carcinoma

The antibody reacted with 2/7 squamous cell carcinomas tested; of theseone was heterogenous in its reactivity.

The two adenocarcinomas were unreactive.

Tissue Distribution

Placenta: All placental tissue, both first trimester and term arenegative. Kidney: Negative Pancreas: Negative Colon: Negative Liver:Negative Endometrium: Negative Buccal cavity: Parabasal cells andstratified epithelium associated with tonsils are positive. Epidermis:Parabasal cells are positive. (foreskin)Biochemical

The target epitope was probed on immunoblots from detergent-solublisedextracts and membrane preparations of enzyme disaggregated cervicalepithelial tissue. The antibody detected a 200 KDa structure undernon-reducing conditions only. The target epitope is therefore likely tobe present on a conformationally-dependent, non-cytokeratin, protein.

EXAMPLE 5 Assessment of Cervical Cells; Comparison with PAP Test

The work described in the preceding examples has resulted in thegeneration of a panel of antibodies with reactivities against epithelialcell populations of the human cervix. The reactivities generallyoverlap, with the exception of Mab 2C7 which reacts specifically withcolumnar cells. The overlapping specificities of the other fourantibodies (6B5, BC4, 9G5 and HG3) demonstrate a continuum ofreactivities that parallel the differentiation lineage of cervicalsquamous epithelial cells themselves. It is most probable that theantibody target epitopes are normal differentiation markers which areexpressed and lost as the cells differentiate from basal to terminalsuperficial squames.

The data herein suggests a scenario wherein with the onset ofpremalignant (CIN) or neoplastic disease conditions, cells can becomearrested at a particular stage of differentiation. This may result in anexpansion of a particular cell population expressing a detectable cellmarker. For example, the anti-parabasal cell reactivity of Mab 6B5 isexpanded in specimens with severe CIN II/III.

Similarly, a pathological condition may result in an expanded cellpopulation accompanied by the concomitant loss of a characteristic cellmarker. For example, the reactivity of Mab BC4, which also reactsagainst parabasal cells in normal epithelium, is reduced in CIN samples.

The reactivities of Mabs 9G5 and HG3 against intermediate andsuperficial squames is markedly reduced in premalignant CIN samples.This could be due to a reduction in the absolute number of intermediateand superficial squames, as a consequence of arrested differentiation atthe parabasal stage. Conversely, it could be due to a loss of therelevant cell markers themselves, as a result of the pathologicalcondition.

It should be noted that an actual expansion or reduction in the absolutenumbers of a particular cell population is not a pre-requisite for thepurposes of the present invention. Any detectable change in antibodybinding relative to established parameters of normality is relevant andof use in the present invention. No particular theory or hypothesislimits the nature and scope of the present invention.

A data bank of numerical parameters pertaining to normal smearsestablishes the “confines of normality”, against which test samples maybe compared. Any significant variance from the established parametersindicates a need for individual diagnosis by suitably qualifiedpersonnel, to assess the clinical status, ie suspect samples arehighlighted using the present invention for further examination.

Experimental Results

Reactivity of the antibodies 9G5, HG3, 6B5, BC4 and 2C7 was assessed onnormal cervical smear samples and CIN-2/CIN-3 cervical smear samples,which were also analysed by the PAP test. The results are shown inTables 3 and 4.

Normal cervical smear material was collected in parallel withpathological smear samples in a colposcopy clinic. Following thedeposition of the primary smear on a glass slide for Papanicolaoustaining, the sampling device, either spatula or brush, was placed in 10mls of Hanks buffered saline and agitated. Samples showing obviouscontamination with blood were discarded. The resulting cell suspensionswere then washed ×2 in this buffer and used to generate cytospins.

Each cytospin spot contained approximately 10⁴ cells.

The MAb reactivities were detected by an indirect immunostainingtechnique using a Streptavidin-Biotin, alkaline phosphatase detectionsystem. The chromogen (Fuschin; Dakopatts) produced a red stain. Cellnuclei were counterstained with Mayer's haematoxylin.

Scoring of % cells stained was determined by counting dispersed cells;total cells stained red/total no. nuclei. Cell clumps are often presentin such preparations and the reactivity of the Mab with these isindicated separately.

The status of the smear sample, normal or CIN, is given as designated bycytological examination (PAP) of the parallel specimen.

Table 3 shows that a pattern of normality for binding of the fivemonoclonal antibodies can be perceived. In the CIN samples binding ofthe same antibodies deviates from this pattern of normality, as shown inTable 4.

It should be noted that in the case of specimens 6 and 7, visually thesesamples appeared similar to normal smears. On morphological grounds, themajority of cells were obviously superficial squames and dyskaryoticcells were not apparent. In other specimens, for example, 1, 2, 3 and10, dyskaryotic cells were clearly evident.

Thus, one can establish a pattern of normality for binding of a panel ofantibodies to a sample containing cells of the cervix such thatdeviation from that pattern in binding of those antibodies to a testsample is indicative of some abnormality which warrants furtherinvestigation.

All documents mentioned herein are incorporated by reference.

REFERENCES

-   Auersperg N and Hauvryl A P, J Nat Cancer Inst 28: pp 605-627, 1962.-   Auersperg N, J Nat Cancer Inst 43: pp 151-173, 1969.-   Harlow E and Lane D (Eds), Antibodies: A Laboratory Manual, Cold    Spring Harbor Laboratory, 1988.-   Holmes C H et al, J Immunology 144:, pp 3099-3105, 1990.-   Houlihan et al, J Immunology 149: pp 668-675, 1992.-   Houlihan J M, PhD Thesis, University of Bristol, 1993.-   Kennet R et al (Eds), Monoclonal Antibodies and T-Cell Hybridomas    (Plenum Press, 1980).-   Knapp W (Ed), Leukocyte Typing IV, Academic Press, London, 1989.-   Laemmli U K, Nature 227: pp 133-681.-   Marchalonis J J, Biochem J. 113: pp 299-305.-   Markwell, Analyt. Biochem. 125; pp 427-432.-   Schrier M et al, Hybridoma Techniques, Cold Spring Harbour    Laboratory, 1080.-   Work and Work (Eds), Laboratory Techniques in Biochemistry and    Molecular Biology, Elsevier, 1979.

TABLE 1 SUMMARY OF MONOCLONAL ANTIBODY REACTIVITIES AGAINST NORMALCERVICAL EPITHELIAL CELLS IN BIOPSY PROVEN NORMAL CERVICES. Target CellMonoclonal Antibodies Populations 2C7 6B5 BC4 9G5 HG3 Basal − +/− − − −Parabasal − + + +/− − Intermediate − − +/− + + Squames Superficial − −− + + Squames Columnar + + − − −

TABLE 2 REACTIVITY OF MABS 9G3 AND HG3 ON CERVICAL CARCINOMAS TumourType 2C7 6B5 BC4 9G5 HG3 A Squamous − +++ − +/− − B Squamous − − − − +++C Squamous − +++ − +/− +++ D Squamous − + − − − E Squamous − + +/− +/−+/− F Squamous − +/− − − +++ G Squamous − − +/− +/− +/− H Adenoc. +++ +− − +++ I Adenoc. − + − − + Squamous = Squamous Cell Carcinoma Adenoc. =Adenocarcinoma +/− Heterogenous reactivity defined as areas of bothreactivity and unreactivity within a specimen. + Positive Reactivity.+++ Extensive intense reactivity.

TABLE 3 REACTIVITY OF MONOCLONAL ANTIBODIES WITH NORMAL CERVICAL SMEARS.(Squamous) (Parabasal) (Columnar) Specimen 9G5¹ HG3¹ 6B5² BC4² 2C7² 1.75% 88% −ve −ve −ve 2. 68% 86% −ve −ve −ve 3. 65% 70% −ve −ve >50   4.75% 85% −ve −ve −ve 5. 62% 79% −ve −ve −ve 6. 93% 68% 5 1 −ve 7. 83% 74%2 −ve >100    8. 72% 85% 7 3 +ve^($) 9. 68% 90% −ve −ve 10 10. 85% 90%−ve −ve −ve mean 74.6 ± 9.79 81.5 ± 8.22 Footnotes ¹200 cells werecounted in the case of 9G5 and HG3. ²Numbers given refer to positivecells in the whole field containing approx. 10⁴ cells. ^($)Clumps ofstained cells were observed.

TABLE 4 REACTIVITY OF MONOCLONAL ANTIBODIES WITH CERVICAL SMEARS FROMPRE-MALIGNANT SPECIMENS WITH CIN-2/CIN-3 (Squamous) (Parabasal)(Columnar) Patient Grade 9G5¹ HG3¹ 6B5² BC4² 2C7² 1. CIN-3 <5% <2% −ve−ve +ve^($) 2. CIN-3 NT <5% −ve −ve +ve^($) 3. CIN-3 <20%   ND >50^($)ve  10^($) 4. CIN-3 60% 20% −ve −ve  1 5. CIN-3 50% 96% −ve −ve −ve 6.CIN-3 75% 73% 10 5 −ve 7. CIN-3 86% 78% ve −ve 10 8. CIN-2/3 54%<10%   >20 2^($) −ve 9. CIN-2/3 59% 52% ve −ve 20 10. CIN-2 15% 15% 19−ve 4% 11. CIN-2 38%^($) 20% vs −ve  1 Footnotes ¹200 cells were countedin the case of 9G5 and HG3. ²Numbers given refer to positive cells inthe whole field containing approx. 10⁴ cells. ^($)Clumps of stainedcells were observed.

1. A method of screening for a premalignant or neoplastic disease statein the squamous cells of a cervical smear sample containing columnar andsquamous cells of the cervix, the method comprising contacting saidsample with a panel of two or more monoclonal antibodies, said panel ofantibodies including at least one monoclonal antibody specific forcolumnar cells and at least one monoclonal antibody specific forsquamous cells, wherein said panel of monoclonal antibodies binds tosurface antigens of normal columnar and squamous cells; verifying thatthe cervical sample comprises columnar cells by detecting the binding ofthe monoclonal antibody specific for columnar cells in the cervicalsample; comparing the pattern of binding of the panel of monoclonalantibodies in said sample with the pattern of binding of said monoclonalantibody panel to a normal cervical cell sample, wherein an alterationof the pattern of binding of the monoclonal antibody or antibodies whichbind to squamous cells in the cervical smear sample relative to thepattern of binding of the monoclonal antibody or antibodies which bindto squamous cells in a normal cervical cell sample is indicative of apremalignant or neoplastic disease state.
 2. A method of screening for apremalignant or neoplastic disease state in the squamous cells of acervical smear sample containing columnar and squamous cells of thecervix, the method comprising contacting said sample with a panel of twoor more monoclonal antibodies, said panel of antibodies including atleast one monoclonal antibody specific for columnar cells and at leastone monoclonal antibody specific for squamous cells, wherein said panelof monoclonal antibodies binds to surface antigens of normal columnarand squamous cells; verifying that the cervical sample comprisescolumnar cells by detecting the binding of the monoclonal antibodyspecific for columnar cells in the cervical sample; comparing thepattern of binding of the monoclonal antibody or antibodies which bindto squamous cells in the cervical smear sample with the pattern ofbinding of said monoclonal antibody or antibodies which bind to squamouscells in a normal cervical cell sample, wherein an alteration of thepattern of binding is indicative of a premalignant or neoplastic diseasestate and wherein the panel includes one or more monoclonal antibodiescomprising an antigen binding domain obtainable from a hybridomaselected from those deposited at the European Collection of Animal CellCultures (ECACC), under the accession numbers ECACC 95020718, ECACC95020716, ECACC 95020720, ECACC 95020717 and ECACC
 95020719. 3. A methodaccording to claim 1 wherein one or more of the monoclonal antibodiesbind to an antigen which can be bound by one or more antibodies obtainedfrom a hybridoma selected from those deposited at the EuropeanCollection of Animal Cell Cultures (ECACC), under the accession numbersECACC 95020718: ECACC 95020716: ECACC 95020720, ECACC 95020717 and ECACC95020719.
 4. A hybridoma selected from those deposited at the EuropeanCollection of Animal Cell Cultures (ECACC), under the accession numbersECACC 95020718, ECACC 95020716, ECACC 95020720, ECACC 95020717 and ECACC95020719.
 5. A monoclonal antibody which specifically binds to thesurface or normal squamous or columnar cells or the cervix comprising anantigen binding domain obtainable from a hybridoma selected from thosedeposited at the European Collection of Animal Cell Cultures (ECACC),under the accession numbers ECACC 95020718, ECACC 95020716, ECACC95020720, ECACC 95020717 and ECACC
 95020719. 6. The method as claimed inclaim 1 wherein said panel of monoclonal antibodies comprises amonoclonal antibody having an antigen binding domain obtainable from ahybridoma deposited at the European Collection of Animal Cell Cultures(ECACC) under the accession number ECACC
 95020716. 7. The method asclaimed in claim 2 wherein said panel includes a monoclonal antibodycomprising an antigen binding domain obtained from the hybridomadeposited under Accession Number ECACC 95020716.